Developments in two separate areas of research may be of interest. The first area relates to methods of monitoring the impact of contaminants on the environment. In a second and separate area of technology, molecular biologists have investigated the genetic regulation of peptide synthesis, utilizing as a model the hs response of a variety of species.
In the first area of research, several kinds of approaches have been used to monitor the impacts of man's activities on the environment. Environmental monitoring has most often focused on evaluating the fate of contaminants through exhaustive chemical analysis of sediment, water, and tissue samples to determine the degree of anthropogenic inputs at a particular site. Correlations have been found between levels of contaminants and the degree of industrial activities in surrounding areas; however, this approach is extremely expensive and gives little insight concerning the effects of these contaminants on organisms in the ecosystem.
In an attempt to address the issue of biological effects, the Environmental Protection Agency ("EPA") has developed water quality criteria that provide a basis for estimating the relative toxicity of each pollutant. In this approach, laboratory bioassay data is obtained for each chemical, and an elaborate procedure is used to derive a criterion number for each chemical that would hypothetically protect organisms in the actual field environment. Although extrapolating from the laboratory to the field has questionable validity, this process does provide a concentration for each pollutant against which the chemical data obtained from a site can be compared. The development of a separate criterion number for each pollutant, however, is a time-consuming and expensive process and is not practical for the over 20,000 pollutants estimated by the EPA to be impacting our waters. This chemical-by-chemical approach also does not take into account the complex interactions that may occur in the complex mixtures of pollutants normally found in the real world.
Because of these problems, the EPA has begun to use short-term bioassays to assess the relative toxicity of complex effluents and surface waters. Gradually, these tests are carried out in the laboratory with a standard organism, such as the fat head minnow or daphnia. The results of these tests tend to be variable, and it is difficult to relate observed toxicity to the presence of pollutants in the test water, let along individual pollutants in a complex mixture. A major problem with these bioassays, as well as with other bioassays, is that it is difficult to extrapolate from laboratory tests to the actual conditions encountered by indigenous organisms in the field.
In another approach, it has bee noted that contaminant-induced stress responses have been observed at every level of biological organization. Initially, contaminants interact with biological systems at the molecular level. These interactions may result in physiological perturbations at the cellular and organismal levels. These perturbations can, in turn, result in effects that have significance at the population and community levels, such as reductions in reproduction, growth, and survival. The time period for expression of toxic effects at the community level is highly variable; it may be on the order of years. Alternatively, where community stability is regulated by one of several "keystone species," subtle stress-induced changes in the reproductive success of these species may be profound, with correspondingly rapid effects on the community and population.
The variations in response time from initial molecular impacts to population and community effects often make it difficult to link inputs of sublethal concentrations of contaminants with perturbations at these higher levels of organization. These problems are further compounded by the natural variability in community structure and productivity that are difficult to differentiate from contaminant-induced changes. For these reasons, cause and effect relationships between contaminant action and biological responses can best be established by examining the mechanisms of contaminant impact with individual organisms. The data can then be related to population and community impacts.
Accordingly, another approach to monitoring involves an initial screening of field sites to determine whether organisms are adversely impacted in situ. With this strategy, one can systematically follow a series of biological and chemical procedures arranged in hierarchical order with appropriate feedback loops to evaluate the extent of impact of stress at the organismal level. The final step involves the identification of specific contaminants that may be the causative agents. This monitoring strategy strives to evaluate, in situ, general stress in the organism and to delineate the specific factors that are involved in a systematic and cost effective fashion. Evaluating stress in native organisms in situ is important because the effect of each environmental variable is dependent on all other variables encountered by the organism. Although organisms may be exposed to a wide range of environmental stressors at any given time, they have only a finite capacity to adapt. Therefore, they integrate the effects of each variable into a total stress load. The effects of subsequent environmental changes will ultimately depend upon the overall exposure history of the organism.
Organisms use many strategies to minimize detrimental effects of environmental changes: they elicit avoidance responses, evoke repair or stabilization mechanisms, and synthesize detoxication enzymes or binding ligands. All of these processes require energy, which is diverted from other cellular processes. As the stress load increases, there will be a threshold at which this diversion disturbs important processes such as growth and reproduction. Thus, perturbations in growth and reproduction have been used as an indicator of stress and are described in Widdows in Mar. Poll. Bull., 16:129-134 (1985). Other types of monitoring techniques that are currently being used as indicators of stress at the organismal level include: scope for growth (Warren and Davis, The Biological Basis of Freshwater Fish Production, Blackwell Scientific, Oxford, pp. 175-214, (1975)), growth inhibition (Sanders and Jenkins, Bio. Bull., 167:704-712 (1984)), and perturbations in the regulation of growth and hormesis (Stebbing, J. Mar. Biol. Assay U.K., 61:35-63 (1981a); Laughlin, Science, 211:705-705 (1981); Sanders, Crustacean Issues, Vol. 2: Crustacean Growth, Ed Wenner, Balkema Press (1985)). However, these techniques are of limited use because: (1) they are not based on the mechanisms that underlie the relationship between general stress physiology and toxicity and, thus, measure stress indirectly; (2) they lack the sensitivity of cellular level parameters; and, (3) they cannot be used conveniently to measure stress in native organisms exposed in situ.
A tiered approach for monitoring biological damage due to contaminant exposure which is rapid and relatively inexpensive would be a major improvement in the state of the art. In this tiered initial screening (tier I) would evaluate the organism's integrated "stress load" as an index of general stress. Negative results at this stage would indicate the organism was not stressed and further testing would be unnecessary. A positive result would require identification of the causation agents. Tier II assays would be undertaken to identify exposure to specific pollutants.
One such approach for identifying the nature of the stressor has been measuring the concentrations of total contaminants in tissues of stressed organisms. However, the relationships between contaminant concentration in organism tissue and toxic effects are complex and difficult to establish because organisms have specific metabolic mechanisms that modify, sequester, compartmentalize, and excrete contaminants. The potential toxicity of a contaminant will depend upon both the amount that has been accumulated and how effectively the organism can metabolize it.
Traditional whole organism techniques for evaluating stress (e.g., growth and reproduction studies) are expensive, time-consuming, and difficult to apply in the field. In addition, these techniques are often organism-specific and cannot easily be applied to a range of localities. As a result of these limitations, a number of biochemical assays have been and are being developed to address this issue.
Most of the biochemical approaches in current use are contaminant-specific in that they only respond to specific class on contaminants (tier II approaches) and, thus, are not useful indicators of general stress. Stressor-specific assays include: (1) the cholinesterase assay in which the activity of the enzyme cholinesterase is used to screen for exposure to organophosphate or carbamate compounds; (2) the mixed function oxidase enzyme (MFO) assay in which induction of synthesis of isoforms of MFOs are used as an indicator of exposure to xenobiotic compounds, including aromatic hydrocarbons and halogenated biphenyls; and, (3) the induction of synthesis and accumulation of metals on the protein, metallothionein, which serves as an indicator of metal exposure.
Not only is each of these methods limited as to the types of contaminants it responds to, but they deal only with exposure and do not necessarily assess the level of stress the organism is experiencing as a consequence of that exposure. Only two biochemical methods are now in use as nonspecific indicators of stress (tier I). One depends upon measuring RNA/DNA ratios that reflect shifts between cell division (DNA synthesis) and nondivision (RNA synthesis) events. The other method makes use of taurine/glycine ratios which reflect metabolic shifts. Both of these ratios are indirect measures of stress, vary in response to metabolic changes that are not stress related, and have not proven useful as sensitive general stress indices.
In view of these shortcomings, the limitations of prior art procedures for evaluating environmental stress are readily apparent. In summary, the prior techniques for addressing environmental stress are all indirect in nature and thus provide ambiguous results. The four major techniques are: (1) direct chemical determinations of contaminants in the environment, (2) total concentration of chemical constituents in tissues of organisms collected from the environment, (3) biological surveys of population and community structures, and, (4) physiological monitoring in laboratory bioassays. Direct chemical measurements in the environment are very expensive and provide limited insight into biological effects of those chemicals on organisms. Measurements of chemical constituents in tissues of organisms correlate poorly with general physiological stress. The high degree of natural variability in biological populations and communities makes it difficult, if not impossible, to establish cause and effect relationships between contaminant exposure and community stress. Laboratory bioassays are usually conducted on single organisms and are limited to individual chemical stressors. They are cumbersome and expensive and do not realistically reflect the complex contaminant mixtures normally encountered in the natural environment.
Accordingly, there is a need for sensitive, rapid, and direct methods for evaluating the total stress load in organisms exposed in the laboratory and in situ. In particular, what is required is a simple and inexpensive field technique that can specifically evaluate the integrated stress load of indigenous organisms and thereby monitor adverse biological effects from contaminant exposure.
The second area of background technology relates to heat shock proteins. These proteins are commonly referred to as heat-shock proteins (hsp's) or heat-stress proteins since it was under conditions of hyperthermia that their synthesis was first observed.
The hsp's are induced by a wide variety of environmental conditions including high levels of heavy metals (Hammond, G. L., Y. K. Lai, C. L. Market, Proc. Natl. Acad. Sci. U.S.A. 79:3485-3488 (1982), Caltabiano, M. M., T. P. Koestler, G. Poste, R. G. Greig, J. Biol. Chem. 261:13381-13386 (1986)), xenobiotics (Irby, R. B., T. W. Snell, B. J. Cochrane, submitted), oxidative compounds (Kapoor, M., J. Lewis, Can. J. Microbiol. 33:162-168 (1987), teratogens (Bournias-Vardiabasis, N., R. L. Teplitz, G. F. Chernoff, R. L. Seecof., Teratology 28:109-122 (1983), Bournias-Vardiabasis, N., C. H. Buzin, Teratogen. Carcinogen. Mutagen. 6:523-536 (1986)), hepatocarcinogens (Carr, B. I., T. H. Huang, C. H. Buzin, K. Itakura, Cancer Res. 46:5106-5111 (1986), anoxia (Spector, M. P., Z. Aliabadi, T. Gonzalez, J. W. Foster, J. Bacteriol. 168:420-424 (1986), and fluctuations in salinity (Ramagopal, S., Plant Physiol. 84:324-331 (1987)).
The heat shock protein response has been observed in bacteria, yeast, plants, Dictyostelium, Tetrahymena, fruit flies, nematodes, chickens, rats, mice, and humans. The response, in fact, has been observed in every species examined to date and, in the case of higher eukaryotes, is not restricted to a particular tissue. See, for example, Neidhardt, Ann. Rev. Genet., 18:295-329 (1984).
Although the number of hsp's induced by heat shock and their exact size are both tissue and species specific, five "universal" hsp's are found in all eukaryotes. Four of these are referred to by their apparent molecular weight on SDS-polyacrylamide gels: hsp 90, hsp 70, hsp 58 and the low molecular weight hsp 20-30. The fifth hsp is an 8 kDa protein called ubiquitin. In eukaryotes each hsp is comprised of a multigene family, the members of which are regulated by different promoters and code for closely related protein isoforms (Lindquist, S., Ann. Rev. Biochem. 55:1151-1191 (1986, Schlesinger, M. J., J. Cell. Biol. 103:321-325 (1986), Schlesinger, M. J., Atlas of Sci. Biochem. 161-164 (1988)). Most of these proteins are synthesized at high levels in stressed cells. However, with the exception of the 72 kDa protein, a highly inducible member of the hsp 70 family, all of these proteins are also present in much lower concentrations in unstressed cells. The initial observations that many hsp's are found in "normal" cells and that hsp 20-30 are developmentally induced in larval systems lead to the suggestion early on that hsp's play a role in normal cellular activities.
Collectively the hsp's appear to be involved in the protection, enhanced survival and restoration of normal cellular activities in stressed cells (Subject, J. R., T.-T. Shyy., Cell. Physiol. 19:C1-C17 (1986). The induction of hsp's by a mild heat shock enhances the tolerance of the cell to subsequent, more severe heat shock, a phenomenon often referred to as thermotolerance or when other environmental conditions are involved "acquired tolerance" (Dean, R. L., B. G. Atkinson, Can. J. Biochem. Cell Biol. 61:472-492 (1982), Landry, J., Bernier, D., Chretien, P., Nicole, L. M., Tanguay, R. M., Marceau, N., Cancer Res. 42:2457-2461 (1982), Berger, H. M., M. P. Woodward, Exp. Cell Res. 147:437-442 (1983), Stephanous, G., S. N. Alahiotis, C. Christogoulou, V. J. Marmaras, Devel. Genet. 299-308 (1983), Roberts, P. B., Int. J. Radiat. Biol. 45:27-31 (1984), Mirkes, P. E., Dev. Biol. 119:115-122 (1987). The induction, expression and decay of acquired tolerance correlates with the induction, accumulation and degradation of heat shock proteins (Landry, J., Bernier, D., Chretien, P., Nicole, L. M., Tanguay, R. M., Marceau, N., Cancer Res. 42:2457-2461 (1982), Subject, J. R., J. Sciandra, R. J., Johnson, Br. J. Radiol 55:579-584 (1983), Nickells R. W., L. W. Browder, Dev. Biol. 112:391-395 (1985), Tomasovic, S. P., T. M. Koval, Int. J. Radiat. Biol. 48:635-650 (1985), Mosser, D. D., J. van Oostrom, N. C. Bols., J. Cell. Physiol. 132:155-160 (1987), Mosser, D. D., N. C. Bols., J. Comp. Physiol. B. 158 (1988).
Upon exposure to a stressor, three distinct events result in a rapid change in metabolic activities within the cell: (1) there is increased transcription of heat shock peptide mRNAs, which are then preferentially translocated to the cytoplasm; (2) the transcription of most other mRNAs is suppressed; and (3) the normal translational activities of the ribosomes are disrupted so that hsp's are preferentially translated. The overall result of these events is that the cell rapidly begins synthesizing hsp's and synthesis of other peptides is repressed. No new peptides or RNA synthesis is necessary to activate the transcription of the heat shock peptide genes, indicating that preexisting factors may be involved. Cell type, state of cell differentiation, type of stressor, and the duration and intensity of stress can affect the quantity and quality of a particular suite of hsp's.
Only very recently have cell biologists begun to understand the molecular mechanisms underlying the physiology of stressed cells. Welch, W. J., J. P. Suhan, J. Cell Biol. 103:2035-2053 (1986). Serious attention to this important cellular phenomena has occurred largely because of the discovery that all cells dramatically alter their gene expression in response to changes in environmental conditions. This alteration in transcriptional activity, referred to as the heat shock response (hsr), appears to be an attempt to protect the cell from damage and to repair existing damage. Schlesinger, M. J., M. Ashburner, A. Tissieres, cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory, pp. 1-440 (1982). Changes in gene expression associated with the hsr are extremely rapid and result in the induced synthesis and accumulation of heat shock proteins. Most hsp's are found in low concentrations in all cells where they play a role in normal cellular function. Although the induction of some of these hsp's is independent of the nature of the stressor, while others are quite stressor specific. These experiments have all dealt with environmentally unrealistic stress or conditions; i.e., conditions unlikely to occur in the environment.
Unfortunately, to date little is known about the environmental relevance of the stress response and much research needs to be focused in this area. Much of the research on the hsr has involved exposure of cells in culture to perturbations which are often extreme and unlikely to occur naturally in the environment (Krause, K. W., E. M. Hallberg, R. L. Hallberg, Molec. Cell. Biol. 6:3854-3861 (1986), Heuss-LaRosa, K., R. R. Mayer, J. H. Cherry, Plant Physiol. 85:4-7 (1987), Welch, W. J., L. A. Mizzen, J. Cell Biol. 106:1117-1130 (1988).
Although the heat shock response is well documented in the literature, the work in this area is being conducted by molecular biologists whose principal focus is using the hsr to address basic molecular genetics questions; e.g., the regulation of gene expression. Much of the work focuses on developing and understanding how genes are regulated in eukaryotes. For example, a recent article by Xiao et al., Science, 239:1139-1142 (1988) describes heat shock gene regulation and concludes that through a determination of what turns on the heat shock gene, it may be possible to design better expression vectors for producing large amounts of desired gene products in eukaryotes. Additional work has focused on the molecular mechanisms of heat shock function in the cell. Antibodies and gene probes for hsp's have been used by molecular biologists to isolate hsp specific clones from gene libraries of various species, to characterize the genetic organization of the heat shock genes, and to study hsp regulation and function. In short, prior use of gene probes and antibodies has been focused on basic research in molecular biology and not on developing assay methods for evaluating biological damage caused by environmental contamination.
The technique most frequently used involves metabolic labeling wherein tissues are incubated with an amino acid tagged with a radioisotope (i.e. .sup.35 S, .sup.14 C, .sup.3 H). The tissue is then homogenized and the proteins are separated by one or two dimensional electrophoresis, and autoradiographed to examine incorporation of the radioisotope into specific proteins. This technique provides information on the entire translational profile in response to a stressor and can be particularly useful for identifying new inducible proteins. However, under continuous exposure to moderate (e.g. sublethal) stress conditions these dramatic changes in translational patterns are transient (approximately 18 hours in Mytilus exposed to a mild heat shock) and translational activity reverts to patterns similar to those found in controls. See, for example, Heikkila, J. J. et al., J. Biol. Chem. 257:12000-12005 (1982); Canvalho, M. and M. S. Freitas, J. Cell. Phys. 137:455-461 (1988); Lindquist, S., Ann. Rev. Biochem. 55:1151-1191 (1986); and Kapoor, M., Int. J. Biochem, 18:15-29 (1986). The short, transient response is followed by a rapid return to control levels of hsp synthesis. Based on these observations using metabolic labeling studies, it would not have been expected that it would not be possible to monitor biological damage to organisms exposed to contaminants in their environment by measuring hsp levels after the initial transient hsp response.
Using methods different from metabolic labelling studies to monitor hsp levels in an organism, the present inventors have surprisingly discovered that the levels of some hsp's remain relatively high in the organism even after they determine the levels of transcription and translation of the hsp's have returned back to control levels.
FIG. 1 herein depicts the type of results obtained from metabolic labeling studies which are used to measure synthetic rates.
FIG. 2 depicts the type of results observed by measuring the concentration of an hsp and its mRNA. These results are the basis of the present assay method.
Accordingly, an object of the present invention is to provide assays and kits for detecting chronic, sublethal environmental contamination by pollutants, and assays and kits for detecting biological damage at the organismal level based on correlating the concentration of heat shock proteins to physiological indices of impairment of the organism.
The manner in which these and other objects are realized by the present invention will be apparent from the summary and detailed description set forth below.